Separation systems with charge mosaic membrane

ABSTRACT

An ion is eluted from an ion exchange resin ( 132 ) in a separation system ( 100 ) using an eluent generated by electrolysis of a medium. Elution is further assisted by an electrical field between two electrodes ( 120, 110 ), wherein the ion exchange resin ( 132 ) is at least partially disposed between the electrodes. Particularly preferred aspects of such separation systems include gradient separation and buffered electrodialysis.

FIELD OF THE INVENTION

The field of the invention is electrophoresis-assisted separation ofionic species.

BACKGROUND OF THE INVENTION

Numerous disciplines in science and technology require separation and/oranalysis of complex mixtures, or quantification, concentration, and/orremoval of various analytes from such mixtures and there are variousseparation technologies known in the art.

For example, individual analytes can be separated or isolated frommixtures using molecular weight differences between the analyte and theremaining compounds in the mixture. Size discrimination may be performedby size exclusion (e.g., using microporous matrix) or by molecularsieving (e.g., using crosslinked matrix). While separations based onmolecular weight differences are typically relatively independent onbuffer conditions and other extraneous factors, resolution betweenanalytes will often become increasingly problematic as the molecularweight difference decreases.

In another example, individual analytes can be separated or isolatedfrom mixtures using differences in hydrophobicity between the analyteand the remaining compounds in the mixture. Numerous separation systemsthat employ such differences are known in the art, and among othersystems, reversed phase high performance liquid chromatography (HPLC)affords a relatively high resolution among relatively chemically similarcompounds. However, many of such systems are difficult to operate whenthe volume of the sample is relatively large (e.g., several liters).Furthermore, HPLC systems are relatively expensive and frequentlyrequire extensive maintenance.

Alternatively, individual analytes can be separated or isolated frommixtures using differences in their net charge at a particular pH and/orionic strength in the sample. Typically such systems include a cationexchange material or an anion exchange material to which one or moreanalytes are bound and eluted using an external elution reagent. Ionexchange separation is a relatively common separation technology that isin many cases inexpensive and frequently has a desirable resolution.However, various difficulaties remain. Among other things, elution of abound analyte will place the analyte in an environment that may not becompatible with further use or that may even interfere with theanalyte's integrity of function.

Still further, analytes may be separated or isolated from mixtures usingdifferences in their affinity towards a typically immobilized and highlyspecific binding agent. Such affinity chromatographic separations aregenerally highly specific and frequently allow gentle separation of theanalyte from the binding agent. However, many affinity reagents arerelatively expensive (e.g., monoclonal antibodies) or may not beavailable for a desired analyte.

In still further known systems, two or more physico-chemical propertiesof an analyte are employed for separation of the analyte from a mixtureof compounds. For example, isoelectric focusing combines pH-dependentvariability of an analyte with electric mobility of the analyte in anelectrophoresis-type of separation. In another example, gelelectrophoresis employs molecular weight and electric charge of anelectrolyte. While many of the separation systems improve at least someaspects of resolution of a desired analyte, various problems stillremain. For example, analyte recovery is frequently problematic.Furthermore, large scale preparation of analytes is often impracticable.Thus, despite various known configurations and methods for separation ofan analyte from a medium, all or almost all suffer from variousproblems. Therefore, there is still a need to provide improvedconfigurations and methods for separation systems.

SUMMARY OF THE INVENTION

The present invention is directed to configurations and methods of aseparation system in which an analyte in ionic form is eluted from anion exchange resin using an electric field and an eluent, wherein theelectric field and the eluent are generated by a pair of electrodes inthe system.

In one aspect of the inventive subject matter, contemplated systemscomprise a cathode, an anode, and a first ion (e.g., anion) bound by afirst ion exchange resin (e.g., anion exchange resin) that is at leastpartially disposed between the cathode and the anode and that isseparated from at least one of the anode and cathode (e.g., cathode) bya charge mosaic membrane (CMM), wherein the cathode, the anode, and theion exchange resin are at least partially disposed in a medium, andwherein the first ion detaches from the ion exchange resin at (a) aparticular voltage applied between the anode and cathode and (b) aparticular electroactivity of a second ion (e.g., hydroxyl ion)generated by electrolysis of the medium (e.g., water).

Particularly contemplated systems comprise a second ion exchange resin(e.g., cation exchange resin) at least partially disposed between theanode and the first ion exchange resin, wherein a cation exchangemembrane is at least partially disposed between the first and second ionexchange resin.

Thus, viewed from another perspective, contemplated systems may comprisean ion exchange resin that binds an ion from a fluid, wherein the ion iseluted from the resin using (a) an electric field generated between ancathode and a anode and (b) a second ion that is generated byelectrolysis of the fluid by the cathode and the anode. In such systems,it is preferred that a charge mosaic membrane separates the ion exchangeresin from the cathode, thereby allowing migration of OH⁻ ions from thecathode to the ion exchange resin and migration of cations from the ionexchange resin to the cathode. While it is generally contemplated thatall or almost all ions may be eluted from the resin using the electricalfield and/or the eluent, additional eluents (e.g., a third ion) may beemployed.

In a further aspect of the inventive subject matter, contemplatedsystems may be employed to separate multiple components from a samplefor analytical or preparative purposes. Consequently, suitable systemsmay include a third ion that binds to the ion exchange resin, whereinthe third ion elutes at an electric field and concentration of thesecond ion that is different from the elution of the ion from the fluid.Especially contemplated fluids and/or media include crude, partiallypurified and/or highly purified preparations/isolates from varioussources, including (bio)synthetic fluids, biological fluids, wastefluids, etc.

Viewed from yet another perspective, contemplated systems may include acharge mosaic membrane coupled to an ion exchange resin that binds anion from a fluid and wherein the ion is eluted at least in part from theresin using an eluent that is generated by electrolysis of the fluid.

Various objects, features, aspects, and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention, along with theaccompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic view of an exemplary CMM gradient separationsystem.

FIG. 2 is a schematic view of an exemplary CMM buffered electrodialysissystem.

FIG. 3 is a schematic view of an exemplary CMM buffered electrodialysissystem.

FIG. 4 is a schematic view of an exemplary CMM gradient electrophoresissystem.

DETAILED DESCRIPTION

The inventors have discovered that ions may be selectively eluted froman ion exchange resin using an electric field and an eluent, wherein theelectric field and the eluent are generated by electrodes that areproximal to ion exchange resin.

More specifically, the inventors discovered that a separation system maycomprise a charge mosaic membrane coupled to an ion exchange resin thatbinds an ion from a fluid and wherein the ion is eluted at least in partfrom the resin using an eluent that is generated by electrolysis of thefluid. Viewed from another perspective, contemplated separation systemsmay include an ion exchange resin that binds an ion from a fluid,wherein the ion is eluted from the resin using (a) an electric fieldgenerated between an cathode and a anode and (b) a second ion that isgenerated by electrolysis of the fluid by the cathode and the anode.

In a particularly preferred configuration, a separation system has acathode, an anode, and a first ion bound by a first ion exchange resinthat is at least partially disposed between the cathode and the anodeand separated from at least one of the anode and cathode by a chargemosaic membrane, wherein the cathode, the anode, and the ion exchangeresin are at least partially disposed in a medium, and wherein the firstion is eluted from the resin using (a) a particular voltage that isapplied between the anode and cathode and (b) a second ion that isgenerated by electrolysis of the medium and moves from the cathode tothe anode by the electric field generated by the particular voltage.

As used herein, the terms “ion bound by an ion exchange resin” and “ionexchange resin that binds an ion” refer to non-covalent, ionic bindingbetween the ion and an ionic or polar group of the ion exchange resin.Consequently, the terms “the ion is eluted from the resin” and “the ionelutes from the resin” refer to breaking of the non-covalent, ionic bondbetween the ion and an ionic or polar group of the ion exchange resin,wherein the breaking of the bond may be effected by (a) an electricfield force that attracts the ion towards an electrode with oppositepolarity, (b) competition for the ionic or polar group of the ionexchange resin by another ion (same type of ion at higher concentrationand/or different ion), and/or (c) kinetic forces acting on the ion(e.g., heat, molecular collisions, etc.).

As also used herein, the term “disposed between the cathode and theanode” refers to a position that intersects or coincides with part of atleast one of a plurality of straight lines between the cathode and theanode. Similarly, the term “disposed between the cathode (or anode) andthe first ion exchange resin” refers to a position that intersects orcoincides with part of at least one of a plurality of straight linesbetween the cathode (or anode) and the first ion exchange resin.

As still further used herein, the term “charge mosaic membrane” refersto a membrane or other support that includes a plurality of chargedgroups, wherein some of the charged groups are positively charged (e.g.,quaternary ammonium groups), wherein other groups are negatively charged(e.g., sulfonic acid groups), and wherein the plurality of chargedgroups are disposed in the membrane or other support such that selectedcations and anions (e.g., H⁺ and OH⁻) can penetrate the membrane orother support while blocking transport of solvent and/or other ions(e.g., proteins with MW of about 30,000 Dalton).

In an especially preferred aspect of the inventive subject matter, anexemplary separation system is configured to operate as a CMM-gradientseparation system. Here, as depicted in FIG. 1 a CMM separation system100 has a housing 102 that at least partially encloses an anodecompartment 120A with anode 120, a cathode compartment 110A with cathode110, and an analyte compartment 130 that is separated from the anodecompartment 120A via cation exchange membrane 180 and that is separatedfrom the cathode compartment 110A via charge mosaic membrane 150. Theanode compartment 120A further includes cation exchange resin 132, whilethe analyte compartment 130A and the cathode compartment 110A includeanion exchange resin 130 and 134, respectively.

Anode, cathode, and analyte compartment further include a mediumcomprising water 160. At least a portion of the water is electrolyzedvia the anode and cathode, wherein oxygen evolves in the anodecompartment, hydrogen evolves in the cathode compartment, and wherein H⁺is generated in the anode compartment and OH⁻ is generated in thecathode compartment. The protons generated in the anode compartment willbe (via cation exchange resin and cation exchange membrane) transportedto the analyte compartment and further (via charge mosaic membrane) tothe cathode compartment. Similarly, the OH⁻ ions generated in thecathode compartment will be transported (via anion exchange resin andcharge mosaic membrane) into the analyte compartment comprising anionexchange resin. However, further passage of the OH⁻ ions to the anodecompartment is blocked by the cation exchange membrane.

A sample comprising ionic species X₁ ⁻Y₁ ⁺ and X₂ ⁻Y₂ ⁺ is applied tothe analyte compartment, and the anionic portions of the sample X₁ ⁻(e.g., first ion 140) and X₂ ⁻ will be bound to the anion exchange resinin the analyte compartment. Upon application of a electric potentialbetween the anode and the cathode, an electric force will act upon thebound anions. Furthermore, electrolysis of water by the electrodes willproved OH⁻ anions that will move from the cathode compartment via theanion exchange resin to the analyte compartment. Thus, an increasingelectrical potential between the electrodes will act in at least twoways upon the anions bound to the anion exchange resin in the analytecompartment. First, an electrophoretic force will increasingly move thebound anions according to their strength with which they bind to theanion exchange material. Second, the OH⁻ ions in the analyticcompartment will increasingly compete for interaction with the anionexchange resin. Consequently, it should be recognized that a particularanion will elute from the anion exchange resin by (a) generation of (andcompetition with) an anion that is generated from the medium byelectrolysis, and (b) at a particular voltage applied to the anode andcathode (via an electrophoretic effect). Additionally, elution mayfurther be assisted by competition with a further anion (as used inconventional ion exchange chromatography).

Alternatively, as depicted in FIG. 2, an exemplary separation system isconfigured to operate as a CMM-buffered electrodialysis system. Here,the separation system 200 has a housing 202 that at least partiallyencloses cathode 210 and anode 220. The housing cooperates with chargemosaic membranes 250 to define a anode compartment 220A, an analytecompartment 230A, and a cathode compartment 210A. The anode compartment220A is at least partially filled with cation exchange resin 232 whilethe cathode compartment 210A is at least partially filled with anionexchange resin 232. The analyte compartment includes an ordered mixedbed comprising alternate layers of cation exchange resin 230A and anionexchange resin 230A′.

Anode, cathode, and analyte compartment further include a mediumcomprising water 260. At least a portion of the water is electrolyzedvia the anode and cathode, wherein oxygen evolves in the anodecompartment, hydrogen evolves in the cathode compartment, and wherein H⁺is generated in the anode compartment and OH⁻ is generated in thecathode compartment. The protons generated in the anode compartment willbe (via cation exchange resin and cation exchange membrane) transportedto the analyte compartment and further (via cation exchange resin andcharge mosaic membrane) to the cathode compartment. Similarly, the OH⁻ions generated in the cathode compartment will be transported (via anionexchange resin and charge mosaic membrane) into the analyte compartmentcomprising anion exchange resin, and further (via anion exchange resinand charge mosaic membrane) to the cathode compartment.

A sample comprising ionic species X⁻Y⁺ and A⁻B⁺ is applied to theanalyte compartment, and the anionic portions of the sample X⁻ and A⁻will be bound to the anion exchange resin in the analyte compartment.Similarly, the cationic portions of the sample Y⁺ and B⁺ will be boundto the cation exchange resin in the analyte compartment. Uponapplication of an electric potential between the anode and the cathode,an electric force will act upon the bound anions and cations.Furthermore, electrolysis of water by the electrodes will provide OH⁻anions and protons that will move from the electrode of their origin tothe electrode with opposite polarity.

Thus, an increasing electrical potential between the electrodes will actin at least two ways upon the anions and cations bound to the anion andcation exchange resin in the analyte compartment. First, anelectrophoretic force will increasingly move the bound anions cationsaccording to their strength with which they bind to the ion exchangematerial. Second, the OH⁻ ions and protons in the analytic compartmentwill increasingly compete for interaction with the anion exchange resinas the electric field strength increases. Consequently, it should berecognized that a particular anion and a particular cation will elutefrom the ion exchange resin by (a) generation of (and competition with)an anion and cation that is generated from the medium by electrolysis,and (b) at a particular voltage applied to the anode and cathode (via anelectrophoretic effect). Thus, it should be further recognized that ionswill typically elute as ion pairs (hence the term ‘buffered CMMelectrodialysis’) from the ion exchange resin. Additionally, elution mayfurther be assisted by competition with a further anions and/or cations(as used in conventional ion exchange chromatography).

With respect to the housing, it is contemplated that the size,configuration and material may vary considerably, and a particularhousing will typically be determined at least in part by the particularfunction of the device and type of sample. However, it is generallycontemplated that the housing is configured to at least partiallyenclose the cathode compartment, the analytical compartment, and/or theanode compartment. Furthermore, suitable housings typically enclose atleast part of the electrodes (which may also be integral part of thehousing). Moreover, it is generally preferred that the materials for thehousing (or at least the materials contacting the anode, analyte, andcathode compartment are chemically and electrically inert (i.e., do notreact with a desired analyte and/or solvent and have a resistivity of atleast 1 MOhm).

Consequently, suitable housings may be fabricated from numerousmaterials, and contemplated materials include natural and syntheticpolymers, metals, glass, and all reasonable combinations thereof.Furthermore, where contemplated devices are employed to isolate one ormore analytes from a relatively large volume (e.g., several liters toseveral hundred liters, and even more), the housing may be configured asa tank, and the separation may be performed batch-wise. On the otherhand, where a continuous flow of sample is preferred, the housing may beconfigured as a column (i.e., generally cylindrical with open ends).

Similarly, contemplated electrodes may be manufactured from a variety ofmaterials, and it is generally contemplated that the particular natureof an electrode will at least partially depend on the particular sample,size of the electrode, and/or strength of the electric field. However,in especially preferred aspects of the inventive subject matter,suitable electrodes include platinum, or platinum-coated electrodes,gold, or gold-coated electrodes, silver or silver-coated electrodes,graphite electrodes, etc.

With respect to the size and positioning of suitable electrodes, it isgenerally preferred that the electrodes are sized and positioned suchthat (a) the electric field between the electrodes overlaps at least inpart with the analyte compartment, and (b) that the electrodes contactthe medium such that at least part of the OH⁻ ions generated by thecathode will migrate towards the anode (preferably at least across theCMM membrane into the analyte compartment). Thus, suitable electrodesmay be configured as wires, plates, grids, or even as integral parts ofthe housing. Furthermore, it should be appreciated that while in somepreferred configurations the electrodes are juxtaposed at the sameheight, other electrode configurations include those in which the heightof one electrode is offset relative to the other electrode.Consequently, the electric field across the analyte compartment incontemplated configurations may be perpendicular relative to the housingand/or CMM membrane, or at an angle between about 1 degree and 89degrees, more typically between 15 degrees and 75 degrees, and mosttypically between 30 degrees and 65 degrees.

Moreover, it should be recognized that contemplated systems may includemultiple electrodes, wherein at least some of the electrodes willprovide for electrolysis of the medium (preferably electrolysis ofwater), while other electrodes may form the electric field in which atleast part of the analyte compartment is disposed.

There are numerous cation and anion exchange resins for the anode,cathode, and analyte compartments known in the art, and it iscontemplated that all known resins are suitable for use in conjunctionwith the teachings presented herein. An exemplary selection of suitableresins is described, for example, in “Ion Chromatography” by James S.Fritz, Douglas T. Gjerde, in “Ion Exchange: Theory and Practice (RoyalSociety of Chemistry Paperbacks)” by C. E. Harland (Springer Verlag;ISBN: 0851864848), or in “Ion-Exchange Sorption and PreparativeChromatography of Biologically Active Molecules (MacromolecularCompounds)” by G. V. Samsonov (Consultants Bureau; ISBN: 0306109883).

Particularly contemplated exchange resins for the anode and cathodecompartment include those with relatively high stability towardsreduction and oxidation of the functional groups under conditionsrequired to separate a desired analyte from a sample fluid, andespecially preferred resins for the anode and cathode compartment have arelatively high capacity for H⁺/OH⁻ exchange. Furthermore, it isgenerally preferred that suitable resins are in solid phase and that atleast some of the resin is in contact with the respective electrode.

With respect to a particular cation/anion exchange resin for the analytecompartment, it is generally contemplated that all resins are especiallysuitable that will bind a desired analyte. There are numerous types ofanion and cation exchange resins with various binding strengths known inthe art, and it is contemplated a person of ordinary skill in the artwill readily identify a particular resin suitable for the analytewithout undue experimentation. Furthermore, and especially where theanalyte is an amphoteric molecule it should be appreciated that the pHof the fluid containing the analyte may be adjusted according to aparticular ion exchange resin.

In still further contemplated aspects, and especially where contemplateddevices are configured as CMM buffered electrodialysis devices, theanalyte compartment may include both anion and cation exchange resins.While not limiting to the inventive subject matter, it is generallypreferred that where the analyte compartment comprises cation and anionexchange resins, the resins are arranged in an ordered sequence. Forexample, suitable sequences include multiple layers of resins in which acation exchange resin alternates with an anion exchange resin, andwherein at least some of the resins extend across the entire width ofthe analyte compartment.

Suitable charge-mosaic membranes may be prepared using numerous methodswell known in the art. For example, cation exchange resins may becombined with anion exchange resins using a polystyrene binder (seee.g., U.S. Pat. No. 2,987,472) or a silicone resin (see e.g., J. N.Weinstein et al., Desalination, 12, 1(1973)). Alternatively, suitablemembranes may also be fabricated by casting or blending polymer phases(see e.g., J. Shorr et al., Desalination, 14, 11(1974) or JapaneseLaid-Open Specification No. 14389/1979). Still further suitable methodsinclude ionotropic-gel membrane methods (see e.g., H. J. Purz, J. Polym.Sci., Part C, 38, 405(1972)), latex-polymer electrolyte methods (seee.g., Japanese Laid-Open Specification No. 18482/1978), or blockcopolymerization methods (see e.g., Y. Isono et al., Macromolecules, 16,1(1983)). In yet further contemplated methods, a cationic, anionic, orneutral polymer may be derivatized to include positive and negativecharges suitable for ion exchange.

Where cationic and anionic polymers are employed to form a charge mosaicmembrane, cationic polymers preferably include primary, secondary ortertiary amino groups, quaternary ammonium groups, or salts thereof,while anionic polymers preferably include sulfonic groups, carboxylicgroups or salts thereof Suitable cationic polymers includepolyvinylpyridine and quaternized products thereof;poly(2-hydroxy-3-methacryloyloxypropyltrimethylammonium chloride);poly(dimethylaminoethyl methacrylate), poly(diethylaminoethylmethacrylate), and copolymers with other monomers and/or polymers.Suitable anionic polymers includepoly-(2-acryloylamino-2-methyl-1-propanesulfonic acid),poly(2-acryloylamino-2-propanesulfonic acid),polymethacryloyloxypropylsulfonic acid, polysulfopropyl methacrylate,poly(2-sulfoethyl methacrylate), polyinylsulfonic acid, polyacrylicacid, polystyrene-maleic acid copolymers, and copolymers with othermonomers and/or polymers.

Furthermore, at least one of the cationic and anionic polymers may becrosslinked using crosslinkers well known in the art. Among numerousalternative crosslinkers, contemplated crosslinkers includedivinylbenzene, methylenebisacrylamide, ethylene glycol dimethacrylateand 1,3-butylene glycol dimethacrylate as well as tri- ortetra-functional acrylates and methacrylates. Still further contemplatedcharge mosaic membranes include those described in U.S. Pat. No.4,976,860 to Takahashi and U.S. Pat. No. 5,304,307 to Linder et al.

In still further contemplated aspects of the inventive subject matter,it should be recognized that suitable membranes may also be configuredto be permeable for charged and non-charged molecules depending on themolecular weight. For example, in such membranes, permeability may beachieved by imparting nanoporosity into the membrane using technologieswell known to the person of ordinary skill in the art. Thus, suitableCMM membranes may be a barrier for molecules with various molecularweights, and it is generally contemplated that a particular degree ofporosity will predominantly determine the molecular weight cut-offcharacteristics of such membranes. For example, where relatively smallpores are formed in the membrane, suitable molecular weight cut-off maybe in the range of between about 300 Da to 3,000 Da. Where somewhatlarger porosity is generated, the molecular weight cut-off may be in therange of between about 3,000 Da to 50,000 Da, and where relatively largepores arte generated, the molecular weight cut-off may be in the rangeof between about 50,000 Da to 200,000 Da, and even higher.

Similarly, it should be recognized that appropriate cation exchangemembranes that separate the anode compartment from the analytecompartment include all or almost all of the known cation exchangemembranes. However, suitable cation exchange membranes especiallyinclude solid polymer electrolyte (SPE) membranes with a relatively highpermeability for protons and a relatively low permeability for solvent.There are numerous SPE membranes known in the art, and various aspectsof exemplary SPE membranes are described, for example, in U.S. Pat. No.3,528,858 to Hodgdon et al, U.S. Pat. No. 3,282,875 to Connolly et al,U.S. Pat. No. 5,635,041 to Bahar et al, and U.S. Pat. No. 5,422,411 toWei et al.

In a further aspect of the inventive subject matter, the origin andcomposition of contemplated samples may vary considerably, and it isgenerally contemplated that the origin and composition of the sample isnot limiting to the inventive subject matter. However, contemplatedsamples are preferably processed or unprocessed biological fluids andespecially include ionic species of polynucleotides, polypeptides,charged lipids, and/or charged carbohydrates. Thus, contemplated samplesmay be prepared or isolated from cell cultures, virus and bacterialcultures, animals (and particularly human), plants and/or fungi.Alternatively, suitable samples also include samples from isolated oropen environments. For example, samples from isolated environmentsinclude process fluids from processing plants (pharmaceutical, food,etc.) while fluids from an open environment may include water samplesfrom a river or other body of water, air, etc.

Furthermore, it should be appreciated that the sample may be providedfor various purposes. Among other things, a sample may be provided toremove, reduce the concentration, or determine presence of one or moreanalytes. Thus, suitable samples may include water, run-off from aprocess, etc. On the other hand, samples may also be provided to isolateor concentrate one or more analytes. Consequently, suitable samples mayinclude biological fluids, chromatographic preparations, etc.

It is generally preferred, however, that preferred samples include waterto at least some degree, and where a particular sample has a relativelylow water content (e.g., less than 10 vol %), it is contemplated thatthe sample may be subjected to a sample preparation step to provide ahigher water content. Furthermore, it should be recognized thatcontemplated samples may be processed to exhibit a particular pH. Forexample, where an analyte is known to have a neutral charge at a firstpH, the pH may be adjusted to an alkaline pH with an appropriate base tofacilitate binding of the analyte to the anion exchange resin in theanalyte compartment.

Consequently, contemplated analytes will particularly include those thatwill exhibit an electric charge at a particular pH, and suitableanalytes include inorganic analytes, organic analytes, and biologicalanalytes. For example, inorganic analytes include elemental ions (e.g.,F⁻, Cl⁻, etc.) and organic and/or inorganic complex ions (e.g., nitrate,carbonate, zirconate, etc.). Organic analytes may include aliphatic,aromatic, and other hydrocarbonaceous ionic molecules, while biologicalanalytes may include ionic forms of nucleic acids, peptides,carbohydrates.

Thus, suitable media particularly include aqueous media, however, inalternative aspects, contemplated media may also include one or morewater-miscible or water-immiscible organic solvents. Exemplarycontemplated water-miscible organic solvents include dimethylsulfoxide,dimethylformamide, various alcohols, various esters, etc., whileexemplary contemplated water-immiscible organic solvents include variousaliphatic hydrocarbons, ethers, etc.

With respect to the voltage that is applied to the electrodes, it shouldbe recognized that a particular voltage will typically be determined byvarious parameters, including salinity of the medium, electrolysis ofthe medium, and strength of the non-covalent bond between the analyteand the ion exchange resin in the analyte compartment. Thus, suitablevoltages will typically be in the range of about less than 1 Volt andseveral 100 Volts. However, it is generally preferred that the voltageis between about 1 Volt and about 100 Volt, and more typically between1.4 Volt and about 50 Volt.

CMM Gradient Separation

In one particularly preferred aspect, contemplated configurations asexemplarily depicted in FIG. 1 may be employed in a system in which asample is applied to the analyte compartment, wherein the samplecomprises an analyte anion that will bind to the anion exchange resin inthe analyte compartment. Application of the sample may be performed in abatch-wise manner as well as in a continuous flow manner in an amountthat will not lead to complete saturation of the binding sites in theanion exchange resin. Upon binding of the analyte anion to the anionexchange resin, a voltage is applied to the electrodes such that twoeffects will additively (or even synergistically) elute the analyteanion from the anion exchange resin.

First, the analyte anion will be subjected to the electric field forcebetween the anode and cathode. Second, electrolysis of the medium(typically water) will generate a competing anion (typically OH⁻) at thecathode, wherein the competing anion will migrate towards the anode (viathe anion exchange resin in the cathode compartment and the CMMmembrane). Consequently, an increasing electric field in contemplatedconfigurations will not only provide an increased electrophoretic force,but also (in addition to the generated competing OH⁻ anions) anincreased electroactivity of the competing anion. While not wishing tobe bound by a particular hypothesis or theory, the inventors contemplatethat an increase in the electric field will increase the kinetic forceof the competing anion by increasing the mobility and force ofinteraction of the competing anion with the bond between the anion ofthe analyte and the anion exchange membrane, thereby increasing theelution force of the competing anion. Viewed from another perspective,an increasing electric field may act in a similar manner to an increasein an exogenously added competing ion (the gradient in a traditional ionexchange chromatography). Thus, the inventors contemplate that theelectric field will act as the gradient in this configuration.

Consequently, it should be especially appreciated that elution of theanion of the analyte may take place without addition of an externalanion (i.e., anion not already present in the sample that is applied tothe analyte compartment) that will compete with the analyte anion. Thus,contemplated configurations may not only be employed to separate orconcentrate a desired compound from a complex mixture, but also todesalinate or otherwise clean up a sample. For example, a sample may beapplied to contemplated separation systems and once the analyte hasbound, the buffer or (other solvent) may be exchanged for another bufferor solvent. Elution of the analyte anion into the new buffer of solventwill then be performed by increase of the electrical field between theanode and cathode (effectively only water via recombination of H⁺ andOH⁻ will be added to the analyte compartment). However, in alternativeaspects of the inventive subject matter, it should also be recognizedthat elution of the analyte anion may further be assisted by addition ofexternal anions to the analyte compartment.

Moreover, it should be recognized that various anions in a sample mayexhibit various elution characteristics (i.e., a first anion is elutedat a first potential between anode and cathode, while a second anion iseluted at a second potential between anode and cathode). Thus,contemplated configurations may also be employed to separate multipleanionic components from a complex mixture by virtue of their inherentelution characteristics at a particular voltage between anode andcathode. In fact, it is even contemplated that such systems may not onlyresolve chemically distinct molecules in a separation, but may alsoresolve stereoisomers of the same compound by virtue of asymmetriccharge distribution in the stereoisomers.

In yet further aspects of the inventive subject matter, it iscontemplated that not only anions may be separated, concentrated, orisolated from a complex mixture, but that contemplated configurationsmay also be employed for cation separation, concentration, and/orisolation. In such configurations, the polarity and arrangement of theion exchange resins, ion exchange membrane, and the CMM membrane areinverted.

CMM Buffered Electrodialysis

In another especially preferred aspect, contemplated configurations asexemplarily depicted in FIG. 2 may be employed in a system in which acomplex sample is applied to the analyte compartment, wherein the samplecomprises a plurality of analyte anions and a plurality of analytecations that will bind to the respective ion exchange resins in theanalyte compartment. Application of the sample may be performed in abatch-wise manner as well as in a continuous flow manner in an amountthat will not lead to complete saturation of the binding sites in theanion and cation exchange resins. Upon binding of the analyte ions tothe ion exchange resins, a voltage is applied to the electrodes suchthat two effects will additively (or even synergistically) elute theanalyte anion from the anion exchange resin.

First, the analyte anion will be subjected to the electric field forcebetween the anode and cathode. Second, electrolysis of the medium(typically water) will generate a competing anion (typically OH⁻) at thecathode, wherein the competing anion will migrate towards the anode (viathe anion exchange resin in the cathode compartment and the CMMmembrane). Consequently, an increasing electric field in contemplatedconfigurations will not only provide an increased electrophoretic force,but also (in addition to the generated competing OH⁻ anions) anincreased electroactivity of the competing anion. Similarly, the analytecation will first be subjected to the electric field force between theanode and cathode. Second, electrolysis of the medium (typically water)will generate a competing cation (typically H⁺) at the anode, whereinthe competing cation will migrate towards the cathode (via the cationexchange resin in the anode compartment and the CMM membrane).Consequently, an increasing electric field in contemplatedconfigurations will not only provide an increased electrophoretic force,but also (in addition to the generated competing H⁺/OH⁻ anions) anincreased electroactivity of the competing cation and anion.

Thus, it should be especially appreciated that (a) the analyte ions in asample will be eluted in an ion pair (hence the term ‘bufferedelectrodialysis’), and that (b) the bound cation and anion will beeluted from their respective resins by an eluent generated byelectrolysis of the medium. (Again, effectively only water viarecombination of H⁺ and OH⁻ will be added to the analyte compartment).However, in alternative aspects of the inventive subject matter, itshould also be recognized that elution of the analyte anion may furtherbe assisted by addition of external anions to the analyte compartment.With respect to the separation, concentration, and isolation aspects ofcontemplated CMM buffered electrodialysis, the same considerations asdescribed above apply.

EXAMPLES

The following examples are provided to further illustrate the inventivesubject matter, and especially to provide further guidance to apractitioner with respect to CMM gradient separation and CMM bufferedelectrodialysis.

CMM Buffered Electrodialysis

Device description: Basic elements of CMM buffered electrodialysis areshown in FIG. 3. All ion-exchange materials are made similar to CMMGradient Separation device. 1-Anode, 2-Anion-exchange screen, 3-Chargemosaic membrane, 4-Cathode, 5-Cation-exchange screen, 6-Bi-chargescreen. The screen is prepared by thermally stitching 4 mm wide stripsof cation- and anion-exchange screens together. Dimensions 5 cm wide and20 cm high. 7-Anode compartment water flow ports, 8-Cathode compartmentwater flow ports, 9-Sample port, 10-Analyte port.

Continuous Operation Mode: This mode can be used for water desalinationand/or demineralization or for separation inorganic and organic (highmolecular weight) component of the solution. Molecular weight cut-off ofthe organic component of the solution is limited by porosity ofcharge-mosaic membranes.

Start water flow through anode and cathode compartments using ports 7A&B and cathode compartment using ports 8 A&B at flow rate of 1 mL/min.

Electric potential between anode and cathode is high enough to flowelectric current at density of 25-to 50-mA/cm2

After period of stabilization, sample is starting to flow at rate of 50mL/min. If sample contains only inorganic component, fraction of thedesalted water can be rerouted and flow through anode and cathodecompartments. Otherwise, the high molecular weight component would beeluted and flown out through port 10.

Periodic (batch) Operation Mode: This mode can be used to separateinorganic and organic component of separated solution.

Start water flow through anode and cathode compartments using ports 7A&B and cathode compartment using ports 8 A&B at flow rate of 0.5mL/min.

Electric potential between anode and cathode is high enough to flowelectric current at density 1-to 5-mA/cm²

Sample of few hundreds milliliters of sample solution is flown throughport 9 at rate of 2 mL/min. Total concentration of ionic, organiccomponents (for example proteins) has to be lower then totalion-exchange capacity of analytical anion-exchange screen. Inorganiccomponents are removed via charge-mosaic membrane. The organiccomponents of the solution are immobilized on anion- or cation exchangeparts of the bi-charge analytical screen.

After sample run is finished, clean water is starting to flow at rate of1 to 2 mL/min and electric potential is gradually increased.Consecutively all deposited components are eluted and can be collected.

Notice: Flow of all organic, non-ionic components of the sample throughthe device is unaffected.

CMM Gradient Separation (CMM Gradient Electrophoresis)

Device description: The basic elements of CMM Gradient Separation areshown on FIG. 4. 1-Anion-exchange screen in cathode compartment; Graftedpoly(chloromethylstyrene) on polyethylene screen subsequentlyquaternized with trimethylamine. Surface charge density about 0.05-0.15meqiv/cm². 2-Cathode made of corrosion-resistant material;3-Charge-mosaic membrane, separating cathode from analyticalcompartment, made from modified polyethylene (R. Gajek et al., J. Polym.Sci., Polym. Phys. Ed., Vol. 19, 1663-1673 (1981)). 4-Anion-exchangeanalytical screen—same as 1. Dimensions: 1 cm×5 cm×0.2 cm. Totalresistivity of 20-200 ohm. 5-Cation-exchange membrane separating anodefrom analytical compartment—any commercially available strongcation-exchange membrane; 6-Anode made as cathode 2. 7-Cation-exchangescreen in anode compartment—made by grafting of polystyrene onpolyethylene screen and subsequent chlorosulfonation and hydrolysis inNaOH solution. Surface charge density about 0.05-0.15 meqiv/cm²;8-Sample injection port with septum for syringe injection of samples;9-A&B—analytical water ports; 10-A&B—cathode water ports; 11-A&B—anodewater ports; 12-Sample outlet port.

Analytical Mode:

Start water flow through anode compartment using ports 11A & B & andcathode compartment using ports 10A & B at flow rate of 2 mL/min.

Apply electric potential between anode and cathode of 1-2 V.

After period of stabilization, sample of 1.0-50 microL can be injectedthrough port 8.

Depending on the sample composition, electric gradient value andduration has to be established for every analysis. At 100 ohm of totalsystem resistivity at 2 V applied potential, current of 20 mA willproduce water flow of approximately 0.2 mL/min. At low electricpotential (less then 1V) higher water flow can be achieved by flowingadditional amount of water through ports 9A and/or 9B.

Separated components can be analyzed using conductivity and/or UV-VISdetectors or can be directly injected to mass spectrum devices. Thesystem can be used for analytical preparative purposes by collectingseparated component using variety of laboratory equipment.

Cleaning Procedure:

Start to flow water through analytical compartment using port 9B as aninlet and 9A as an outlet at rate of 2-4 mL/min.

Change polarity of electrodes and apply 2-5 V of electric potential.Notice: in such configuration water electrolysis will occur betweencation-exchange membrane (5) and analytical screen (4). Resulting H+ andOH− ions will flow in opposite directions comparing to analytical mode.

Thus, specific embodiments and applications of improved separationsystems with charge mosaic membranes have been disclosed. It should beapparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced.

1. A separation system comprising: a cathode, an anode, and a first ionbound by a first ion exchange resin, wherein the first ion exchangeresin is at least partially disposed between the cathode and the anodeand separated from at least one of the anode and cathode by a chargemosaic membrane; wherein the cathode, the anode, and the ion exchangeresin are at least partially disposed in a medium; and wherein the firstion is eluted from the resin using (a) a voltage that is applied betweenthe anode and cathode and (b) a second ion that is generated byelectrolysis of the medium.
 2. The separation system of claim 1 whereinthe first ion is an anion, the first ion exchange resin is an anionexchange resin that is separated from the cathode by the charge mosaicmembrane, and wherein the second ion is a hydroxyl ion.
 3. Theseparation system of claim 2 wherein the medium comprises water.
 4. Theseparation system of claim 3 wherein the second ion reacts with an H⁺ion generated at the anode to form water.
 5. The separation system ofclaim 1 further comprising a second ion exchange resin at leastpartially disposed between the anode and the first ion exchange resin.6. The separation system of claim 5 further comprising a cation exchangemembrane at least partially disposed between the first and second ionexchange resin.
 7. The separation system of claim 6 wherein the chargemosaic membrane is at least partially disposed between the cathode andthe first ion exchange resin.
 8. A separation system comprising an ionexchange resin that binds an ion from a fluid, wherein the ion is elutedfrom the resin using (a) an electric field generated between an cathodeand a anode and (b) a second ion that is generated by electrolysis ofthe fluid by the cathode and the anode, and wherein a charge mosaicmembrane separates the ion exchange resin from the cathode, therebyallowing migration of OH⁻ ions from the cathode to the ion exchangeresin and migration of cations from the ion exchange resin to thecathode.
 9. (canceled)
 10. The separation system of claim 8 whereinelution of the ion from the fluid further comprises addition of a thirdion.
 11. The separation system of claim 8 wherein the ion exchange resincomprises an anion exchange resin, and wherein the ion is an anion. 12.The separation system of claim 8 wherein the fluid comprises abiological fluid and wherein the ion comprises at least one of apolynucleotide, a polypeptide, a charged lipid, and a chargedcarbohydrate.
 13. The separation system of claim 8 further comprising athird ion that binds to the ion exchange resin, wherein the third ionelutes at an electric field and concentration of the second ion that isdifferent from the elution of the ion from the fluid.
 14. A separationsystem comprising a charge mosaic membrane that is coupled to an ionexchange resin wherein the resin binds an ion from a fluid and whereinthe ion is eluted at least in part from the resin using an eluent thatis generated by electrolysis of the fluid.
 15. The separation system ofclaim 14 wherein the resin comprises an anion ion exchange resin, andwherein the eluent is an OH⁻ ion.
 16. The separation system of claim 14wherein electrolysis is performed by a current applied to an anode and acathode, wherein the anode and the cathode are at least partiallydisposed in the fluid, wherein the resin is at least partially disposedbetween the anode and the cathode.
 17. The separation system of claim 16wherein elution of the ion is assisted by an electrical field generatedbetween the anode and the cathode.
 18. The separation system of claim 14wherein the fluid comprises a biological fluid and wherein the ioncomprises at least one of a polynucleotide, a polypeptide, a chargedlipid, and a charged carbohydrate.
 19. The separation system of claim 14wherein elution is further assisted by addition of a second ion to theion exchange resin.
 20. The separation system of claim 19 wherein thesecond ion is an anion.